Engineering the glutamate transporter homologue GltPh using protein semisynthesis
| Autor: | Francis I. Valiyaveetil, Paul J. Focke, Alvin W. Annen |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
chemistry.chemical_classification
Amino Acid Transport System X-AG Archaeal Proteins Transporter Peptide Protein engineering Native chemical ligation Protein Engineering Biochemistry Semisynthesis Protein Structure Secondary Article Amino acid Substrate Specificity chemistry Native state Peptides Ion transporter |
| Zdroj: | Biochemistry. 54(8) |
| ISSN: | 1520-4995 |
| Popis: | Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins. |
| Databáze: | OpenAIRE |
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